Endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe as well as synthesis method and application thereof

A nitroreductase, fluorescent probe technology, applied in fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., to achieve the effects of high yield, high sensitivity and simple synthesis method

Inactive Publication Date: 2017-12-08
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the current lack of endoplasmic reticulum-targeted two-photon nitroreductase fluorescent prob

Method used

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  • Endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe as well as synthesis method and application thereof
  • Endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe as well as synthesis method and application thereof
  • Endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe as well as synthesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0044] Example 1 Synthesis of fluorescent probe.

[0045] In a 25 mL round-bottom flask, slowly add 1 mmol of ethylenediamine (compound 1) dropwise to toluene (5 mL) at 40 ℃, add 1.0 mmol of p-toluenesulfonyl chloride (compound 2) with sufficient stirring, and heat After refluxing for 3-4 h, cool to room temperature, add 5ml of 2M HCl aqueous solution to the reaction, remove the filter residue by suction filtration, neutralize the filtrate with NaOH until a large amount of white solid precipitates, filter under reduced pressure and vacuum dry the solid to obtain a solid N-(2-aminoethyl)-4-methylbensulfonamide (Compound 3), yield: 72%. The product was directly subjected to the next reaction without purification.

[0046] In a 100 mL round-bottom flask, add 243 mg of 4-nitronaphthalene dicarboxylate (compound 4) (1 mmol) and 20 mL of ethanol, and add 0.1 mL of triethylamine and 321 mg of N-(2-aminoethyl) with stirring. )-4-methylbenzenesulfonamide (compound 3) (1.5 mmol), then heat...

Example Embodiment

[0048] Example 2 Fluorescence probe Na-ER-NTR absorption spectrum test

[0049] The fluorescent probe Na-ER-NTR in Example 1 was weighed and prepared with dimethyl sulfoxide (DMSO) to prepare a 1 mM mother liquor.

[0050] Take 25 μL of the probe mother solution and add them to 3 identical 5 mL volumetric flasks, add 225 μL of DMSO solution, and add reduced coenzyme I (NADH, final concentration 300 μM) to two of them. One of the two volumetric flasks with coenzyme was added with phosphate buffer solution of nitroreductase (final concentration 2.0 μg / mL), and the other was not added. The three volumetric flasks are all fixed to volume with phosphate buffer (0.01M PBS, pH = 7.4), reacted at 37 ℃ for 60 min, and then subjected to the absorption spectrum test. The wavelength is the abscissa and the absorbance is the ordinate. figure 2 . by figure 2 It can be seen that the absorption intensity of a single probe is very small, and a weak absorption peak appears at 360 nm. After addin...

Example Embodiment

[0051] Example 3 Kinetic test of the interaction between fluorescent probe Na-ER-NTR and nitroreductase.

[0052] The fluorescent probe mother solution in Example 2 was diluted to 5 μM probe working solution for later use.

[0053] The nitroreductase was prepared with a concentration of 0, 0.5, 1.0, 1.5, 2.5, and 5.0 μg / mL in PBS with a pH of 7.4 and a concentration of 0.01 M.

[0054] The probe working solution was mixed with different concentrations of nitroreductase, the final concentration of NADH was 300μM, and then the fluorescence detection (λ ex = 440 nm, λ em = 545 nm), test every 3 minutes, test for 80 minutes, record the fluorescence intensity of each system over time, and establish a curve of fluorescence intensity over time, such as Figure 4 It is shown that the fluorescence intensity of the reaction system gradually increases, and tends to be stable after nearly 60 minutes, and the fluorescence intensity of the reaction system reaches a saturated state.

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Abstract

The invention provides an endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe, wherein the chemical name is 4-nitro-N-(2-(4-methyl benzenesulfonamido)ethyl)-naphthalimide. The endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe is synthesized by the following steps: enabling ethylenediamine to completely react with paratoluensulfonyl chloride to obtain N-(2-aminoethyl)-4-toluenesulfonamide; enabling 4-nitro-1,8-naphthalic anhydride to react with the N-(2-aminoethyl)-4-toluenesulfonamide to obtain the endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe. The invention also provides application of the fluorescent probe in detecting nitroreductase activity in a solution and a biological body by use of single-proton or two-photon fluorescence. The probe synthesis method provided by the invention is simple and realizes high yield; the probe can be positioned on the endoplasmic reticulum to perform two-photon fluorescence detection on cells or tissues, and also has high sensitivity and can resist the interference of multiple interfering substances.

Description

technical field [0001] The invention relates to a fast-response nitroreductase fluorescent probe, in particular to an endoplasmic reticulum-targeted two-photon effect fluorescent probe and its synthesis method and application, belonging to the field of organic small molecule fluorescent probes. Background technique [0002] Hypoxia is an important feature of the occurrence and development of malignant tumors, mainly due to the unlimited growth and metabolism of tumor cells, which increases oxygen consumption, resulting in insufficient blood oxygen supply in the tumor area. Medical research shows that the tumor hypoxic area generally refers to the cell population in the 10-20 μm area of ​​the tumor blood vessel, and the cells in this area are also called hypoxic cells. Because hypoxic cells do not have sufficient oxygen supply, the cells are in a relatively quiescent state where they cannot proliferate or die, and this state is generally at the boundary between G1 and S phase...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07D221/14G01N21/64
CPCC09K11/06C07D221/14C09K2211/1007C09K2211/1029G01N21/6486
Inventor 林伟英唐永和徐安马燕燕
Owner UNIV OF JINAN
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